Method for preparing dendrobium officinale peptide by enzymolysis method

Abstract

According to the method, contact of dendrobium officinale protein and protease in the enzymolysis process is promoted through an acid solution, the enzymolysis efficiency is improved, meanwhile, the protease is further promoted to enter substrate particles, peptide bonds are broken, and more small molecule peptides are generated; a double bond is introduced to the surface of a konjac polysaccharide carrier through a specific process method, so that polymerization reaction occurs on the surface of the carrier, a flexible cross-linked bridge is constructed between polymer long chains, a cross-linked network is formed, a microenvironment taking an enzyme aggregate as a center is provided for enzymolysis reaction, the problem of insufficient enzymolysis caused by aggregation of dendrobium officinale protein is avoided, and the enzymolysis efficiency is improved. The product yield is improved; the molecular sieve chromatographic column is combined to realize molecular recognition of small differences in structure and physicochemical properties in the mixture after enzymolysis in the separation process, dendrobium officinale small-molecular peptide with small molecular weight is separated in a centralized manner, and the biological activity of the product is improved.

Description

Technical Field

[0001] The invention relates to the technical field of deep processing of traditional Chinese medicines, and in particular to a method for preparing dendrobium officinale peptides by enzymatic hydrolysis. Background Art

[0002] Dendrobium officinale is a traditional Chinese medicine obtained by drying the stems of Dendrobium officinale Kimura et Migo, a plant of the Orchidaceae family. It is known as the "life-saving magic grass" among the Chinese people. Modern pharmacological studies have shown that Dendrobium officinale has pharmacological effects such as lowering blood sugar, enhancing immunity, anti-oxidation, anti-tumor and anti-aging. The leaves of Dendrobium officinale are rich in a variety of proteins needed by the human body, but they need to be broken down into small molecule peptides or amino acids by the body and absorbed by the body before the medicinal effects of Dendrobium officinale can be exerted.

[0003] At present, there are five common preparation methods for peptide compounds, namely enzymatic hydrolysis, chemical synthesis, natural extraction, microbial fermentation and DNA recombination. Among them, enzymatic hydrolysis is a classic method and the main method for preparing small molecule peptides. However, this method has the disadvantage of low production efficiency, and the molecular weight distribution of peptide compounds prepared by enzymatic hydrolysis is wide, resulting in a further decrease in the content of small molecule peptides that can be fully absorbed by the human body to exert their efficacy, and the bioavailability of the product is low. Summary of the invention

[0004] The purpose of the present invention is to avoid the shortcomings of the above-mentioned prior art and provide an enzymatic method for preparing Dendrobium officinale peptides with an average molecular weight below 1000D and high bioavailability.

[0005] The above-mentioned object of the present invention is achieved by the following technical scheme: A method for preparing Dendrobium officinale peptide by enzymatic hydrolysis, comprising the following steps: Step 1: drying the Dendrobium officinale leaves in an oven at 60° C., crushing the dried Dendrobium officinale leaves with a powder grinder, and then passing through an 80-mesh sieve to obtain Dendrobium officinale leaf powder; Step 2: add deionized water to the Dendrobium officinale leaf powder at a solid-liquid ratio of 1:25-31, perform ultrasonic treatment for 10 minutes, add an acidic solution 4-8 times the weight of the Dendrobium officinale leaf powder, stir thoroughly and continue ultrasonic treatment for 30 minutes, then centrifuge at 3500-5000 rpm for 30 minutes, take the supernatant and filter it through a 0.22 μm filter membrane to obtain a Dendrobium officinale hydrolyzate, wherein the ultrasonic treatment power is 150-300 W and the frequency is 50-75 kHz; Step 3, take 34-58 parts of konjac flour and evenly disperse them in 100-180 parts of deionized water, stir until the konjac flour fully absorbs water and swells to form konjac sol, add 18-22 parts of benzyl acrolein, stir for 10-20 minutes, adjust the pH value to 10.0-11.0 with sodium hydroxide, keep warm in a water bath at 80-90° C. for 3 hours, stand and cool, then add 30-60 parts of cyclohexyl methanol, soak at 40-65° C. for 24 hours, add 16-23 parts of alkaline protease and stir for 5-6 hours to obtain fixed alkaline protease; Step 4: adding fixed alkaline protease to the hydrolyzed Dendrobium officinale, wherein the amount of the fixed alkaline protease is 0.04-0.08% of the weight of the hydrolyzed Dendrobium officinale; after enzymolysis for 2 hours, inactivating the enzyme in a 90° C. water bath for 30 minutes, cooling, and centrifuging at 1500-2500 rpm for 30 minutes; Step 5: Apply the supernatant after centrifugation to a molecular sieve chromatography column, elute with 0.01 mol / L phosphate buffer at pH 7.2 at a flow rate of 0.05 mL / min, and collect the Dendrobium officinale peptide.

[0006] Preparation of acidic solution: Take 4-8 parts of sodium hypochlorite phosphate and dissolve them in 19-32 parts of deionized water, add 2-6 parts of chromium chloride, stir at 70-110° C. for 1.5 hours to obtain mixture A; take 38-43 parts of hexadecyltrimethylammonium bromide, add phosphoric acid to adjust the pH value to 5.5-6.8, stir and activate at room temperature for 0.5 hours; then add mixture A, mix ultrasonically for 30 minutes, and obtain the acidic solution.

[0007] Preparation method of molecular sieve chromatography column: 1) Take 4-6 parts of methylene bis-nonylphenol polyoxyethylene ether, add 30-40 parts of deionized water and 8-11 parts of 2 mol / L HCl, stir to dissolve, add 8.5-10 parts of triphenyl phosphite, stir for 5-8 hours, dry at 95℃ for 1 day, and calcine in air atmosphere at 480℃ for 6 hours to obtain a molecular sieve precursor; 2) Add 46-57 parts of 10% toluene solution of 2-phenylpropyltrichlorosilane and 5-9 parts of p-nitro-N,N-dimethylaniline to the molecular sieve precursor, stir at 60°C for 20 hours, centrifuge at 1500-3000 r / min for 15 minutes after cooling, wash the precipitate with deionized water and vacuum dry for 48 hours to obtain the molecular sieve; 3) After the molecular sieve is soaked in 0.01 mol / L phosphate buffer at pH=7.2 for 5-8 hours, it is loaded into a chromatography column and equilibrated with the above phosphate buffer to obtain the molecular sieve chromatography column.

[0008] The present invention has the following beneficial effects: (1) Surfactants can bind to lignin through hydrophobic interaction, thereby promoting enzymatic efficiency. The technical solution of the present invention modifies hexadecyltrimethylammonium bromide so that there are more hydrogen bonding sites between the modified surfactant molecules and the hydrolyzed protein of Dendrobium officinale. Compared with general surfactants, it can form a stable chemical bond with the hydrophobic inner cavity of the hydrolyzed protein of Dendrobium officinale, so that the ordered α-helical structure in the secondary structure of the hydrolyzed protein is transformed into an irregular curl, the microscopic surface integrity of the hydrolyzed protein is destroyed, a large number of small spherical particles are attached to the large particles, and the particles become loose. The dense spherical structure of the protein of Dendrobium officinale is opened, and the specific surface area is increased. Due to the increase in the number of pores and small particles on the surface, the surface area of ​​the protein of Dendrobium officinale as a substrate in contact with the hydrolase increases during the enzymatic hydrolysis process, thereby improving its enzymatic efficiency. In addition, the pore structure is also conducive to the hydrolase entering the interior of the substrate particles, breaking the peptide bonds and producing more small molecule peptides.

[0009] (2) The technical solution of the present invention first removes a large amount of hemicellulose in the solution through an acidic solution, and then adsorbs the residual lignin through the modified surfactant therein, thereby avoiding the ineffective adsorption of lignin on the enzyme and weakening the effect of lignin in the solution on the enzymatic hydrolysis, thereby shortening the enzymatic hydrolysis time, saving the amount of enzyme, thereby reducing the enzymatic hydrolysis cost and improving production feasibility. The enzymatic hydrolysis time can be significantly shortened, the enzymatic hydrolysis efficiency can be improved, and the amount of cellulase used can be effectively reduced.

[0010] (3) The alkaline protease prepared by coupling with polysaccharide as a carrier has good catalytic activity and stability in use. However, since the dense spherical structure of Dendrobium officinale protein in the system is opened and the specific surface area increases, the granular and porous Dendrobium officinale protein is prone to agglomeration during the enzymatic hydrolysis process. The immobilized enzyme prepared by conventional methods will lead to an increase in the number of micelles in the system and an increase in the internal volume of the micelles, making it more difficult for the reaction substrate to approach the alkaline protease, thereby hindering the reaction between the reaction substrate and the enzyme on the interface layer.

[0011] (4) Double bonds are introduced on the surface of the konjac polysaccharide carrier by a specific process method, a polymerization reaction occurs on the carrier surface, and a flexible cross-linking bridge is constructed between the long polymer chains to form a cross-linking network, which provides suitable pores and interconnected channels for the adsorption and capture of granular porous proteins, and achieves efficient adsorption of granular porous proteins of Dendrobium officinale. Alkaline protease is loaded on the surface of the modified konjac polysaccharide carrier, and the carrier loaded with alkaline protease is dispersed in the system to realize enzymatic hydrolysis to produce small molecule peptides. When the alkaline protease is loaded on the surface of the modified konjac polysaccharide carrier, the granular porous Dendrobium officinale eggs are driven to preferentially aggregate with the alkaline protease on the surface of the modified konjac polysaccharide carrier, providing a microenvironment centered on the enzyme aggregate for the enzymatic hydrolysis reaction, avoiding the problem of insufficient enzymatic hydrolysis caused by the aggregation of Dendrobium officinale proteins, and improving the product yield.

[0012] (5) Dendrobium officinale peptides with a molecular weight of less than 1000 have good activity and are easy to be absorbed by the human body. Conventional separation methods have a wide molecular weight distribution range and poor biological activity. In addition, compounds with similar structures and physicochemical properties exist in the mixture after enzymatic hydrolysis, which makes separation and purification extremely difficult. Conventional column chromatography has low separation selectivity for natural active substances. In the chromatographic column prepared by the technical solution of the present invention, negatively charged groups are introduced onto the surface of the molecular sieve to regulate the interaction mode and strength between the molecular sieve and the small molecule peptide, so as to realize the molecular recognition of slight differences in structure and physicochemical properties in the mixture after enzymatic hydrolysis during the separation process, and can concentrate on separating small molecule peptides of Dendrobium officinale with smaller molecular weight, thereby improving the biological activity of the product. DETAILED DESCRIPTION Example 1

[0013] Prepare the acid solution: Take 4 parts of sodium hypochlorite phosphate and dissolve them in 32 parts of deionized water, add 6 parts of chromium chloride, stir at 70°C for 1.5 hours to obtain mixture A; take 38 parts of hexadecyltrimethylammonium bromide, add phosphoric acid to adjust the pH value to 6.8, stir and activate at room temperature for 0.5 hours; then add mixture A, mix ultrasonically for 30 minutes, and obtain an acidic solution.

[0014] Preparation of molecular sieve chromatography column: 1) Take 6 parts of methylene bis-nonylphenol polyoxyethylene ether, add 30 parts of deionized water and 11 parts of 2 mol / L HCl, stir to dissolve, add 10 parts of triphenyl phosphite, stir for 8 hours, dry at 95°C for 1 day, and calcine in air atmosphere at 480°C for 6 hours to obtain a molecular sieve precursor; 2) Add 46 parts of 10% toluene solution of 2-phenylpropyltrichlorosilane and 5 parts of p-nitro-N,N-dimethylaniline to the molecular sieve precursor, stir at 60°C for 20 hours, cool and centrifuge at 3000r / min for 15 minutes, wash the precipitate with deionized water and vacuum dry for 48 hours to obtain the molecular sieve; 3) After the molecular sieve is soaked in 0.01 mol / L phosphate buffer at pH = 7.2 for 5 hours, it is loaded into the chromatography column and balanced with the above phosphate buffer to obtain the molecular sieve chromatography column.

[0015] Preparation of Dendrobium officinale peptide: Step 1: drying the Dendrobium officinale leaves in an oven at 60° C., crushing the dried Dendrobium officinale leaves with a powder grinder, and then passing through an 80-mesh sieve to obtain Dendrobium officinale leaf powder; Step 2: add deionized water to the Dendrobium officinale leaf powder at a solid-liquid ratio of 1:25, treat for 10 minutes at an ultrasonic power of 300 W and a frequency of 50 kHz, add an acidic solution 4 times the weight of the Dendrobium officinale leaf powder, stir thoroughly and continue ultrasonic treatment for 30 minutes, then centrifuge at 5000 rpm for 30 minutes, take the supernatant and filter through a 0.22 μm filter membrane to obtain a Dendrobium officinale hydrolyzate; Step 3, take 58 parts of konjac flour and evenly disperse them in 100 parts of deionized water, stir until the konjac flour fully absorbs water and swells to form konjac sol, add 22 parts of benzyl acrolein, stir for 10 minutes, adjust the pH value to 11.0 with sodium hydroxide, keep warm in a 90°C water bath for 3 hours, stand and cool, then add 60 parts of cyclohexyl methanol, soak at 40°C for 24 hours, add 23 parts of alkaline protease and stir for 6 hours to obtain fixed alkaline protease; Step 4: adding 0.08% of the weight of the immobilized alkaline protease to the hydrolyzed Dendrobium officinale solution, enzymolysis for 2 hours, inactivating the enzyme in a 90° C. water bath for 30 minutes, cooling and centrifuging at 1500 rpm for 30 minutes; Step 5: Apply the supernatant after centrifugation to a molecular sieve chromatography column, elute with 0.01 mol / L phosphate buffer at pH 7.2 at a flow rate of 0.05 mL / min, and collect the Dendrobium officinale peptide. Example 2

[0016] Prepare the acid solution: Take 8 parts of sodium hypochlorite phosphate and dissolve it in 19 parts of deionized water, add 2 parts of chromium chloride, stir at 110°C for 1.5 hours to obtain mixture A; take 43 parts of hexadecyltrimethylammonium bromide, add phosphoric acid to adjust the pH value to 5.5, stir and activate at room temperature for 0.5 hours; then add mixture A, mix ultrasonically for 30 minutes, and obtain an acidic solution.

[0017] Preparation of molecular sieve chromatography column: 1) Take 4 parts of methylene bis-nonylphenol polyoxyethylene ether, add 40 parts of deionized water and 8 parts of 2 mol / L HCl, stir to dissolve, then add 8.5 parts of triphenyl phosphite, stir for 5 hours, dry at 95°C for 1 day, and calcine in air atmosphere at 480°C for 6 hours to obtain a molecular sieve precursor; 2) Add 57 parts of 10% toluene solution of 2-phenylpropyltrichlorosilane and 9 parts of p-nitro-N,N-dimethylaniline to the molecular sieve precursor, stir at 60°C for 20 hours, cool and centrifuge at 1500r / min for 15 minutes, wash the precipitate with deionized water and vacuum dry for 48 hours to obtain the molecular sieve; 3) After the molecular sieve is soaked in 0.01 mol / L phosphate buffer at pH = 7.2 for 8 hours, it is loaded into the chromatography column and equilibrated with the above phosphate buffer to obtain the molecular sieve chromatography column.

[0018] Preparation of Dendrobium officinale peptide: Step 1: drying the Dendrobium officinale leaves in an oven at 60° C., crushing the dried Dendrobium officinale leaves with a powder grinder, and then passing through an 80-mesh sieve to obtain Dendrobium officinale leaf powder; Step 2: add deionized water to the Dendrobium officinale leaf powder at a solid-liquid ratio of 1:31, treat for 10 minutes at an ultrasonic power of 150 W and a frequency of 75 kHz, add an acidic solution 8 times the weight of the Dendrobium officinale leaf powder, stir thoroughly and continue ultrasonic treatment for 30 minutes, then centrifuge at 3500 rpm for 30 minutes, take the supernatant and filter through a 0.22 μm filter membrane to obtain a Dendrobium officinale hydrolyzate; Step 3, take 34 parts of konjac flour and evenly disperse them in 180 parts of deionized water, stir until the konjac flour fully absorbs water and swells to form konjac sol, add 18 parts of benzyl acrolein, stir for 30 minutes, adjust the pH value to 10.0 with sodium hydroxide, keep warm in a water bath at 80°C for 3 hours, stand and cool, then add 30 parts of cyclohexyl methanol, soak at 65°C for 24 hours, add 16 parts of alkaline protease and stir for 5 hours to obtain fixed alkaline protease; Step 4: adding 0.04% of the weight of the immobilized alkaline protease to the hydrolyzed Dendrobium officinale solution, enzymolysis for 2 hours, inactivating the enzyme in a 90° C. water bath for 30 minutes, cooling and centrifuging at 2500 rpm for 30 minutes; Step 5: Apply the supernatant after centrifugation to a molecular sieve chromatography column, elute with 0.01 mol / L phosphate buffer at pH 7.2 at a flow rate of 0.05 mL / min, and collect the Dendrobium officinale peptide. Example 3

[0019] Prepare the acid solution: Take 6 parts of sodium hypochlorite phosphate and dissolve them in 25.5 parts of deionized water, add 4 parts of chromium chloride, stir at 90°C for 1.5 hours to obtain mixture A; take 40.5 parts of hexadecyltrimethylammonium bromide, add phosphoric acid to adjust the pH value to 6.2, and activate by stirring at room temperature for 0.5 hours; then add mixture A, mix ultrasonically for 30 minutes, and obtain an acidic solution.

[0020] Preparation of molecular sieve chromatography column: 1) Take 5 parts of methylene bis-nonylphenol polyoxyethylene ether, add 35 parts of deionized water and 9.5 parts of 2 mol / L HCl, stir to dissolve, add 9 parts of triphenyl phosphite, stir for 6.5 hours, dry at 95°C for 1 day, and calcine in air atmosphere at 480°C for 6 hours to obtain a molecular sieve precursor; 2) Add 51.5 parts of 10% toluene solution of 2-phenylpropyltrichlorosilane and 7 parts of p-nitro-N,N-dimethylaniline to the molecular sieve precursor, stir at 60°C for 20 hours, cool and centrifuge at 2300r / min for 15 minutes, wash the precipitate with deionized water and vacuum dry for 48 hours to obtain the molecular sieve; 3) After the molecular sieve is soaked in 0.01 mol / L phosphate buffer at pH = 7.2 for 6.5 hours, it is loaded into the chromatography column and equilibrated with the above phosphate buffer to obtain the molecular sieve chromatography column.

[0021] Preparation of Dendrobium officinale peptide: Step 1: drying the Dendrobium officinale leaves in an oven at 60° C., crushing the dried Dendrobium officinale leaves with a powder grinder, and then passing through an 80-mesh sieve to obtain Dendrobium officinale leaf powder; Step 2: Add deionized water to the Dendrobium officinale leaf powder at a solid-liquid ratio of 1:28, treat for 10 minutes at an ultrasonic power of 220 W and a frequency of 60 kHz, add an acidic solution 6 times the weight of the Dendrobium officinale leaf powder, stir thoroughly and continue ultrasonic treatment for 30 minutes, then centrifuge at 4200 rpm for 30 minutes, take the supernatant and filter through a 0.22 μm filter membrane to obtain a Dendrobium officinale hydrolyzate; Step 3, take 46 parts of konjac flour and evenly disperse them in 140 parts of deionized water, stir until the konjac flour fully absorbs water and swells to form konjac sol, add 20 parts of benzyl acrolein, stir for 20 minutes, adjust the pH value to 10.5 with sodium hydroxide, keep warm in a water bath at 85°C for 3 hours, stand and cool, then add 45 parts of cyclohexyl methanol, soak at 52°C for 24 hours, add 19.5 parts of alkaline protease and stir for 5.5 hours to obtain fixed alkaline protease; Step 4: adding 0.06% of the weight of the immobilized alkaline protease to the hydrolyzed Dendrobium officinale solution, enzymolysis for 2 hours, inactivating the enzyme in a 90° C. water bath for 30 minutes, cooling and centrifuging at 2000 rpm for 30 minutes; Step 5: Apply the supernatant after centrifugation to a molecular sieve chromatography column, elute with 0.01 mol / L phosphate buffer at pH 7.2 at a flow rate of 0.05 mL / min, and collect the Dendrobium officinale peptide.

[0022] Comparative Example 1 This comparative example prepares an acidic solution: Take 6 parts of sodium phosphate and dissolve it in 25.5 parts of deionized water, add 4 parts of chromium chloride, stir at 90°C for 1.5 hours to obtain mixture A; take 40.5 parts of hexadecyltrimethylammonium bromide, add phosphoric acid to adjust the pH value to 6.2, stir and activate at room temperature for 0.5 hours; then add mixture A, mix ultrasonically for 30 minutes, and obtain an acidic solution.

[0023] The rest is the same as in Example 3.

[0024] Comparative Example 2 This comparative example prepares an acidic solution: Take 6 parts of sodium hypochlorite phosphate and dissolve it in 25.5 parts of deionized water, stir at 90°C for 1.5 hours, add 40.5 parts of hexadecyltrimethylammonium bromide, adjust the pH value to 6.2 with phosphoric acid, activate it with stirring at room temperature for 0.5 hours, and mix it ultrasonically for 30 minutes to obtain an acidic solution.

[0025] The rest is the same as in Example 3.

[0026] Comparative Example 3 This comparative example prepares an acidic solution: Take 6 parts of sodium hypochlorite phosphate and dissolve them in 25.5 parts of deionized water, add 4 parts of chromium chloride, stir at 65°C for 1.5 hours to obtain mixture A; take 40.5 parts of hexadecyltrimethylammonium bromide, add phosphoric acid to adjust the pH value to 6.2, stir and activate at room temperature for 0.5 hours; then add mixture A, mix ultrasonically for 30 minutes, and obtain an acidic solution.

[0027] The rest is the same as in Example 3.

[0028] Comparative Example 4 This comparative example prepares an acidic solution: Take 6 parts of sodium hypochlorite phosphate and dissolve them in 25.5 parts of deionized water, add 4 parts of chromium chloride, stir at 115°C for 1.5 hours to obtain mixture A; take 40.5 parts of hexadecyltrimethylammonium bromide, add phosphoric acid to adjust the pH value to 6.2, and activate by stirring at room temperature for 0.5 hours; then add mixture A, mix ultrasonically for 30 minutes, and obtain an acidic solution.

[0029] The rest is the same as in Example 3.

[0030] Comparative Example 5 This comparative example prepares an acidic solution: Take 6 parts of sodium hypochlorite phosphate and dissolve them in 25.5 parts of deionized water, add 4 parts of chromium chloride, stir at 90°C for 1.5 hours to obtain mixture A; take 40.5 parts of hexadecyltrimethylammonium bromide, add phosphoric acid to adjust the pH value to 5.0, stir and activate at room temperature for 0.5 hours; then add mixture A, mix ultrasonically for 30 minutes, and obtain an acidic solution.

[0031] The rest is the same as in Example 3.

[0032] Comparative Example 6 The difference of this comparative example is that a phosphoric acid solution with a pH value of 6.2 is used as the acidic solution, and the rest is the same as that of Example 3.

[0033] Comparative Example 7 This comparative example prepares a molecular sieve chromatography column: 1) Take 5 parts of methylene bis-nonylphenol polyoxyethylene ether, add 35 parts of deionized water and 7 parts of 2 mol / L HCl, stir to dissolve, then add 9 parts of triphenyl phosphite, stir for 6.5 hours, dry at 95°C for 1 day, and calcine in air atmosphere at 480°C for 6 hours to obtain a molecular sieve precursor; 2) Add 51.5 parts of 10% toluene solution of 2-phenylpropyltrichlorosilane and 7 parts of p-nitro-N,N-dimethylaniline to the molecular sieve precursor, stir at 60°C for 20 hours, cool and centrifuge at 2300r / min for 15 minutes, wash the precipitate with deionized water and vacuum dry for 48 hours to obtain the molecular sieve; 3) After the molecular sieve is soaked in 0.01 mol / L phosphate buffer at pH = 7.2 for 6.5 hours, it is loaded into the chromatography column and equilibrated with the above phosphate buffer to obtain the molecular sieve chromatography column.

[0034] The rest is the same as in Example 3.

[0035] Comparative Example 8 This comparative example prepares a molecular sieve chromatography column: 1) Take 5 parts of methylene bis-nonylphenol polyoxyethylene ether, add 35 parts of deionized water and 12 parts of 2 mol / L HCl, stir to dissolve, then add 9 parts of triphenyl phosphite, stir for 6.5 hours, dry at 95°C for 1 day, and calcine in air atmosphere at 480°C for 6 hours to obtain a molecular sieve precursor; 2) Add 51.5 parts of 10% toluene solution of 2-phenylpropyltrichlorosilane and 7 parts of p-nitro-N,N-dimethylaniline to the molecular sieve precursor, stir at 60°C for 20 hours, cool and centrifuge at 2300r / min for 15 minutes, wash the precipitate with deionized water and vacuum dry for 48 hours to obtain the molecular sieve; 3) After the molecular sieve is soaked in 0.01 mol / L phosphate buffer at pH = 7.2 for 6.5 hours, it is loaded into the chromatography column and equilibrated with the above phosphate buffer to obtain the molecular sieve chromatography column.

[0036] The rest is the same as in Example 3.

[0037] Comparative Example 9 This comparative example prepares a molecular sieve chromatography column: 1) Take 5 parts of methylene bis-nonylphenol polyoxyethylene ether, add 35 parts of deionized water and 9.5 parts of 2 mol / L HCl, stir for 6.5 hours, dry at 95°C for 1 day, and calcine in air atmosphere at 480°C for 6 hours to obtain a molecular sieve precursor; 2) Add 51.5 parts of 10% toluene solution of 2-phenylpropyltrichlorosilane and 7 parts of p-nitro-N,N-dimethylaniline to the molecular sieve precursor, stir at 60°C for 20 hours, cool and centrifuge at 2300r / min for 15 minutes, wash the precipitate with deionized water and vacuum dry for 48 hours to obtain the molecular sieve; 3) After the molecular sieve is soaked in 0.01 mol / L phosphate buffer at pH = 7.2 for 6.5 hours, it is loaded into the chromatography column and equilibrated with the above phosphate buffer to obtain the molecular sieve chromatography column.

[0038] The rest is the same as in Example 3.

[0039] Comparative Example 10 This comparative example prepares a molecular sieve chromatography column: 1) Take 5 parts of methylene bis-nonylphenol polyoxyethylene ether, add 35 parts of deionized water and 9.5 parts of 2 mol / L HCl, stir to dissolve, add 9 parts of triphenyl phosphite, stir for 6.5 hours, dry at 95°C for 1 day, and calcine in air atmosphere at 480°C for 6 hours to obtain a molecular sieve precursor; 2) Add 51.5 parts of toluene solution and 7 parts of p-nitro-N,N-dimethylaniline to the molecular sieve precursor, stir at 60°C for 20 hours, cool and centrifuge at 2300r / min for 15 minutes, wash the precipitate with deionized water and vacuum dry for 48 hours to obtain the molecular sieve; 3) After the molecular sieve is soaked in 0.01 mol / L phosphate buffer at pH = 7.2 for 6.5 hours, it is loaded into the chromatography column and equilibrated with the above phosphate buffer to obtain the molecular sieve chromatography column.

[0040] The rest is the same as in Example 3.

[0041] Comparative Example 11 This comparative example prepares a molecular sieve chromatography column: 1) Take 5 parts of methylene bis-nonylphenol polyoxyethylene ether, add 35 parts of deionized water and 9.5 parts of 2 mol / L HCl, stir to dissolve, add 9 parts of triphenyl phosphite, stir for 6.5 hours, dry at 95°C for 1 day, and calcine in air atmosphere at 480°C for 6 hours to obtain a molecular sieve precursor; 2) Add 51.5 parts of 10% toluene solution of 2-phenylpropyltrichlorosilane to the molecular sieve precursor, stir at 60°C for 20 hours, cool and centrifuge at 2300r / min for 15 minutes, wash the precipitate with deionized water and vacuum dry for 48 hours to obtain the molecular sieve; 3) After the molecular sieve is soaked in 0.01 mol / L phosphate buffer at pH = 7.2 for 6.5 hours, it is loaded into the chromatography column and equilibrated with the above phosphate buffer to obtain the molecular sieve chromatography column.

[0042] The rest is the same as in Example 3.

[0043] Comparative Example 12 In the process of preparing Dendrobium officinale peptide in this comparative example, the ultrasonic treatment conditions used in step 2 are different; Step 2: Add deionized water to the Dendrobium officinale leaf powder at a solid-liquid ratio of 1:28, treat for 10 minutes at an ultrasonic power of 100 W and a frequency of 60 kHz, add an acidic solution 6 times the weight of the Dendrobium officinale leaf powder, stir thoroughly and continue ultrasonic treatment for 30 minutes, then centrifuge at 4200 rpm for 30 minutes, and take the supernatant and filter it through a 0.22 μm filter membrane to obtain the Dendrobium officinale hydrolyzate.

[0044] The rest is the same as in Example 3.

[0045] Comparative Example 13 In the process of preparing Dendrobium officinale peptide in this comparative example, the ultrasonic treatment conditions used in step 2 are different; Step 2: Add deionized water to the Dendrobium officinale leaf powder at a solid-liquid ratio of 1:28, treat for 10 minutes with an ultrasonic power of 220 W and a frequency of 40 kHz, add an acidic solution 6 times the weight of the Dendrobium officinale leaf powder, stir thoroughly and continue ultrasonic treatment for 30 minutes, then centrifuge at 4200 rpm for 30 minutes, and take the supernatant and filter it through a 0.22 μm filter membrane to obtain the Dendrobium officinale hydrolyzate.

[0046] The rest is the same as in Example 3.

[0047] Comparative Example 14 This comparative example prepares Dendrobium officinale peptide: Step 1: drying the Dendrobium officinale leaves in an oven at 60° C., crushing the dried Dendrobium officinale leaves with a powder grinder, and then passing through an 80-mesh sieve to obtain Dendrobium officinale leaf powder; Step 2: Add deionized water to the Dendrobium officinale leaf powder at a solid-liquid ratio of 1:28, treat for 10 minutes at an ultrasonic power of 220 W and a frequency of 60 kHz, add an acidic solution 6 times the weight of the Dendrobium officinale leaf powder, stir thoroughly and continue ultrasonic treatment for 30 minutes, then centrifuge at 4200 rpm for 30 minutes, take the supernatant and filter through a 0.22 μm filter membrane to obtain a Dendrobium officinale hydrolyzate; Step 3: adding 0.06% alkaline protease by weight of the Dendrobium officinale hydrolyzate to the Dendrobium officinale hydrolyzate, enzymolyzing for 2 hours, inactivating the enzyme in a 90° C. water bath for 30 minutes, cooling and centrifuging at 2000 rpm for 30 minutes; Step 4: Take the supernatant after centrifugation and apply it to a molecular sieve chromatography column, elute with 0.01 mol / L phosphate buffer at pH=7.2 at a flow rate of 0.05 mL / min to collect Dendrobium officinale peptides.

[0048] The rest is the same as in Example 3.

[0049] Comparative Example 15 In the process of preparing Dendrobium officinale peptide in this comparative example, the conditions for preparing the immobilized alkaline protease in step 3 are different: Step 3: Take 46 parts of konjac flour and evenly disperse them in 140 parts of deionized water, stir until the konjac flour fully absorbs water and swells to form konjac sol, then adjust the pH value to 10.5 with sodium hydroxide, keep warm in a water bath at 85°C for 3h, let stand and cool, then add 45 parts of cyclohexylmethanol, soak at 52°C for 24h, add 19.5 parts of alkaline protease and stir for 5.5h to obtain immobilized alkaline protease.

[0050] The rest is the same as in Example 3.

[0051] Comparative Example 16 In the process of preparing Dendrobium officinale peptide in this comparative example, the conditions for preparing the immobilized alkaline protease in step 3 are different: Step 3: Take 46 parts of konjac flour and evenly disperse them in 140 parts of deionized water, stir until the konjac flour fully absorbs water and swells to form konjac sol, add 20 parts of benzyl acrolein, stir for 20 minutes, adjust the pH value to 9.5 with sodium hydroxide, keep warm in a water bath at 85°C for 3 hours, let stand and cool, then add 45 parts of cyclohexyl methanol, soak at 52°C for 24 hours, add 19.5 parts of alkaline protease and stir for 5.5 hours to obtain fixed alkaline protease.

[0052] The rest is the same as in Example 3.

[0053] Comparative Example 17 In the process of preparing Dendrobium officinale peptide in this comparative example, the conditions for preparing the immobilized alkaline protease in step 3 are different: Step 3: Take 46 parts of konjac flour and evenly disperse them in 140 parts of deionized water, stir until the konjac flour fully absorbs water and swells to form konjac sol, add 20 parts of acrolein, stir for 20 minutes, adjust the pH value to 11.5 with sodium hydroxide, keep warm in a water bath at 85°C for 3 hours, let stand and cool, then add 45 parts of cyclohexylmethanol, soak at 52°C for 24 hours, add 19.5 parts of alkaline protease and stir for 5.5 hours to obtain fixed alkaline protease.

[0054] The rest is the same as in Example 3.

[0055] Comparative Example 18 In the process of preparing Dendrobium officinale peptide in this comparative example, the conditions for preparing the immobilized alkaline protease in step 3 are different: Step 3: Take 46 parts of konjac flour and evenly disperse them in 140 parts of deionized water, stir until the konjac flour fully absorbs water and swells to form konjac sol, add 20 parts of acrolein, stir for 20 minutes, adjust the pH value to 10.5 with sodium hydroxide, keep warm in a 75°C water bath for 3 hours, let stand and cool, then add 45 parts of cyclohexylmethanol, soak at 52°C for 24 hours, add 19.5 parts of alkaline protease and stir for 5.5 hours to obtain fixed alkaline protease.

[0056] The rest is the same as in Example 3.

[0057] Comparative Example 19 In the process of preparing Dendrobium officinale peptide in this comparative example, the conditions for preparing the immobilized alkaline protease in step 3 are different: Step 3: Take 46 parts of konjac flour and evenly disperse them in 140 parts of deionized water, stir until the konjac flour fully absorbs water and swells to form konjac sol, add 20 parts of benzyl acrolein, stir for 20 minutes, adjust the pH value to 10.5 with sodium hydroxide, keep warm in a 95°C water bath for 3 hours, let stand and cool, then add 45 parts of cyclohexyl methanol, soak at 52°C for 24 hours, add 19.5 parts of alkaline protease and stir for 5.5 hours to obtain fixed alkaline protease.

[0058] The rest is the same as in Example 3.

[0059] Comparative Example 20 In the process of preparing Dendrobium officinale peptide in this comparative example, the conditions for preparing the immobilized alkaline protease in step 3 are different: Step 3: Take 46 parts of konjac flour and evenly disperse them in 140 parts of deionized water, stir until the konjac flour fully absorbs water and swells to form konjac sol, add 20 parts of acrolein, stir for 20 minutes, adjust the pH value to 10.5 with sodium hydroxide, keep warm in a water bath at 85°C for 3 hours, let it stand and cool, add 19.5 parts of alkaline protease and stir for 5.5 hours to obtain immobilized alkaline protease.

[0060] The rest is the same as in Example 3.

[0061] Comparative Example 21 In the process of preparing Dendrobium officinale peptide in this comparative example, the amount of fixed alkaline protease added in step 4 is different; Step 4: Add 0.03% of the weight of the fixed alkaline protease to the hydrolyzed Dendrobium officinale solution, enzymolyze for 2 hours, inactivate the enzyme in a 90° C. water bath for 30 minutes, and centrifuge at 2000 rpm for 30 minutes after cooling.

[0062] The rest is the same as in Example 3.

[0063] Comparative Example 22 In the process of preparing Dendrobium officinale peptide in this comparative example, the amount of fixed alkaline protease added in step 4 is different; Step 4: Add 0.09% of the weight of the fixed alkaline protease to the Dendrobium officinale hydrolyzate, enzymolyze for 2 hours, inactivate the enzyme in a 90° C. water bath for 30 minutes, and centrifuge at 2000 rpm for 30 minutes after cooling.

[0064] The rest is the same as in Example 3.

[0065] Performance Testing Determination of degree of hydrolysis (DH) The degree of hydrolysis refers to the percentage of the number of peptide bonds broken during the hydrolysis of a protein to the total number of peptide bonds in the protein. The hydrolyzed Dendrobium officinale solution after enzymatic hydrolysis in the process of preparing Dendrobium officinale peptides was tested, and the free hydrolysis degree of the technical solution of the present invention was determined by formaldehyde titration.

[0066] Determination of peptide mass concentration: The supernatant of the hydrolyzed Dendrobium officinale after enzymatic hydrolysis in the process of preparing Dendrobium officinale peptides was centrifuged and fully mixed with an equal volume of 10 g / 100 mL trichloroacetic acid, allowed to stand at 4 ° C for 30 min, centrifuged at 8000 r / min for 5 min, and the polypeptide content in the supernatant was determined by the Coomassie Brilliant Blue method.

[0067] Determination of peptide molecular weight: The peptide from Dendrobium officinale was diluted to 1 mg / mL with acetonitrile, and the molecular weight distribution of the peptide was determined by gel chromatography. Detection conditions: chromatographic column TSKG3000PwxL chromatographic column 300mm×7.8mm, 10μm; mobile phase V(acetonitrile):V(water):V(trifluoroacetic acid)=45:55:0.1; flow rate: 0.5mL·min-1; column temperature: 30℃; detection wavelength: 220nm; injection volume 10μL.

[0068] The technical indicators of Examples 1 to 3 of the present invention are compared with those of Comparative Examples 1 to 22, as shown in Tables 1 and 2.

[0069] Table 1

[0070] Table 2

[0071] Combining Examples 1 to 3 and Comparative Examples 1 to 6 in Table 1, it can be seen that the acidic solution can promote the contact between the Dendrobium officinale protein and the protease during the enzymolysis process, improve the enzymolysis efficiency, and is also conducive to the protease entering the interior of the substrate particles, breaking the peptide bonds, and producing more small molecule peptides; Combining Examples 1 to 3 and Comparative Examples 12 to 22 in Table 1, it can be seen that the technical solution of the present invention introduces double bonds on the surface of the konjac polysaccharide carrier by a specific process method, a polymerization reaction occurs on the surface of the carrier, and a flexible cross-linking bridge is constructed between the long chains of the polymer to form a cross-linked network, which provides a microenvironment centered on the enzyme aggregate for the enzymolysis reaction, avoids the problem of insufficient enzymolysis caused by the aggregation of the Dendrobium officinale protein, and improves the product yield; Combining Examples 1 to 3 and Comparative Examples 7 to 11 in Table 2, it can be seen that the molecular sieve prepared by the technical solution of the present invention can realize the molecular recognition of the slight differences in the structure and physicochemical properties in the mixture after enzymolysis during the separation process, and concentrates on separating the small molecule peptides of Dendrobium officinale with a smaller molecular weight, thereby improving the biological activity of the product.

Claims

1. A method for preparing Dendrobium officinale peptide by enzymatic hydrolysis, Features: The following steps are involved: Step 1: drying the Dendrobium officinale leaves in an oven at 60° C., crushing the dried Dendrobium officinale leaves with a powder grinder, and then passing through an 80-mesh sieve to obtain Dendrobium officinale leaf powder; Step 2: add deionized water to the Dendrobium officinale leaf powder at a solid-liquid ratio of 1:25-31, perform ultrasonic treatment for 10 minutes, add an acidic solution 4-8 times the weight of the Dendrobium officinale leaf powder, stir thoroughly and continue ultrasonic treatment for 30 minutes, then centrifuge at 3500-5000 rpm for 30 minutes, and filter the supernatant through a 0.22 μm filter membrane to obtain a Dendrobium officinale hydrolyzate; Step 3, take 34-58 parts of konjac flour and evenly disperse them in 100-180 parts of deionized water, stir until the konjac flour fully absorbs water and swells to form konjac sol, add 18-22 parts of benzyl acrolein, stir for 10-20 minutes, adjust the pH value to 10.0-11.0 with sodium hydroxide, keep warm in a water bath at 80-90° C. for 3 hours, stand and cool, then add 30-60 parts of cyclohexyl methanol, soak at 40-65° C. for 24 hours, add 16-23 parts of alkaline protease and stir for 5-6 hours to obtain fixed alkaline protease; Step 4: Add fixed alkaline protease to the hydrolyzed Dendrobium officinale solution, enzymolyze for 2 hours, inactivate the enzyme in a 90°C water bath for 30 minutes, and centrifuge at 1500-2500 rpm for 30 minutes after cooling; Step 5: Apply the supernatant after centrifugation to a molecular sieve chromatography column, elute with 0.01 mol / L phosphate buffer at pH 7.2 at a flow rate of 0.05 mL / min, and collect the Dendrobium officinale peptide.

2. The method for preparing Dendrobium officinale peptide by enzymatic hydrolysis as claimed in claim 1, Features: In step 2, the ultrasonic treatment is performed under the conditions of power 150-300 W and frequency 50-75 kHz.

3. The method for preparing Dendrobium officinale peptide by enzymatic hydrolysis as claimed in claim 1, Features: In step 2, the preparation method of the acidic solution is: Take 4-8 parts of sodium hypochlorite phosphate and dissolve them in 19-32 parts of deionized water, add 2-6 parts of chromium chloride, stir at 70-110° C. for 1.5 hours to obtain mixture A; take 38-43 parts of hexadecyltrimethylammonium bromide, add phosphoric acid to adjust the pH value to 5.5-6.8, stir and activate at room temperature for 0.5 hours; then add mixture A, mix ultrasonically for 30 minutes, and obtain the acidic solution.

4. The method for preparing Dendrobium officinale peptide by enzymatic hydrolysis as claimed in claim 1, Features: In step 4, the amount of fixed alkaline protease added is 0.04-0.08% by weight of the Dendrobium officinale hydrolyzate.

5. The method for preparing Dendrobium officinale peptide by enzymatic hydrolysis as claimed in claim 1, Features: In step 5, the preparation method of the molecular sieve chromatography column is: 1) Take 4-6 parts of methylene bis-nonylphenol polyoxyethylene ether, add 30-40 parts of deionized water and 8-11 parts of 2 mol / L HCl, stir to dissolve, add 8.5-10 parts of triphenyl phosphite, stir for 5-8 hours, dry at 95℃ for 1 day, and calcine in air atmosphere at 480℃ for 6 hours to obtain a molecular sieve precursor; 2) Add 46-57 parts of 10% toluene solution of 2-phenylpropyltrichlorosilane and 5-9 parts of p-nitro-N,N-dimethylaniline to the molecular sieve precursor, stir at 60°C for 20 hours, centrifuge at 1500-3000 r / min for 15 minutes after cooling, wash the precipitate with deionized water and vacuum dry for 48 hours to obtain the molecular sieve; 3) After the molecular sieve is soaked in 0.01 mol / L phosphate buffer at pH=7.2 for 5-8 hours, it is loaded into a chromatography column and equilibrated with the above phosphate buffer to obtain the molecular sieve chromatography column.

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